PROJECT SUMMARY/ABSTRACT Defects in glucose sensing are at the root of a number of metabolic disorders. Impaired regulation of blood glucose levels due to defects in glucose sensing may cause severe metabolic disorders such as diabetes, and many types of cancer cells depend on a high rate of glucose consumption to maintain their viability. Thus, learning how cells sense and respond to glucose is of great interest and major significance. The goal of this proposal is to understand the molecular mechanisms underlying glucose sensing and signaling in the budding yeast Saccharomyces cerevisiae. The yeast senses glucose through the two transporter-like glucose receptors (TLGRs) Rgt2 and Snf3, also known as glucose sensors, which generate an intracellular signal in response to glucose that induces expression of genes involved in glucose uptake and metabolism. However, it is largely unknown how the glucose receptors generate the glucose signal and transduce it across the plasma membrane to the intracellular machinery. It has been speculated that TLGRs signal glucose availability through the plasma membrane-tethered casein kinases Yck1 and Yck2 (YckI, the yeast orthologs of mammalian CK1?), but their role is controversial and their mechanism of activation not well understood. To achieve the goal of this application, we propose two specific aims. Specific Aim 1 of this proposal characterizes the molecular mechanisms of glucose sensing by the glucose receptors at the plasma membrane. TLGRs retain the ability to bind glucose but can no longer transport this sugar; instead they signal glucose availability. Our study indicates that the glucose receptors are removed from the plasma membrane by endocytosis and degraded in the vacuole when glucose is depleted. We propose genetic and bioinformatic approaches to identify TLGR mutations that alter the TLGR-mediated glucose signaling. Specific Aim 2 defines the role of YckI in TLGR signaling. YckI has been suggested as a downstream signal transducer of the glucose reecptors. However, YckI appears to interact with the C-terminal domain of the glucose receptors and positively regulates their function. Using genetic, biochemical, and cell biological approaches, we will assess the role of YckI in TLGR signaling, with a focus on the biological significance of this interaction. Collectively, successful completion of each aim will provide a higher resolution view of glucose sensing at the plasma membrane and help to establish a general paradigm for extracellular glucose sensing in higher eukaryotes, where defects in glucose homeostasis are central to a variety of human pathologies.